Memory B cells anticipate SARS-CoV-2 variants through somatic hypermutation
Authors: Matthias Bruhn, Maureen Obara, Abhishek Chiyyeadu, Bibiana Costa, Abdus Salam, Annett Ziegler, Inken Waltl, Andreas Pavlou, Agnes Bonifacius, Markus Hoffmann, Theresa Graalmann, Stefan Pöhlmann, Britta Eiz-Vesper, Axel Schambach, Ulrich Kalinke
Journal: Journal of Infection (2023)

Dear Editor, Memory B cells (MBC) are an important element of the long-lived immunological memory and they provide a diverse pool of B cell receptors that are utilized rapidly under conditions of re-infection.1Inoue T. Kurosaki T. Memory B cells.Nat Rev Immunol. 2023; (online ahead of print)https://www.nature.com/articles/s41577-023-00897-3.pdfCrossref PubMed Scopus (15) Google Scholar In a recent publication, Mohn et al. monitored spike- and RBD-specific MBC in individuals who recovered from infection with SARS-CoV-2.2Mohn K.G.I. Bredholt G. Onyango T.B. Brokstad K.A. Zhou F. Holter J.C. et al.SARS-CoV-2 infection induces long-lived B and T-cell responses up to 15 months post-infection, irrespective of disease severity.J Infect. 2023; 87: 346-349Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar The authors concluded that irrespective of disease severity, robust and long-lived MBC responses are mounted. They noticed that infection-induced MBC continued to evolve over time with a shift towards RBD-specificity, an effect the authors attributed to affinity maturation.2Mohn K.G.I. Bredholt G. Onyango T.B. Brokstad K.A. Zhou F. Holter J.C. et al.SARS-CoV-2 infection induces long-lived B and T-cell responses up to 15 months post-infection, irrespective of disease severity.J Infect. 2023; 87: 346-349Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar For multiple months after recovery, MBC mature via the introduction of additional somatic hypermutations (SHM) and thereby improve the affinity and binding quality of their antibodies.3Gaebler C. Wang Z. Lorenzi J.C.C. Muecksch F. Finkin S. Tokuyama M. et al.Evolution of antibody immunity to SARS-CoV-2.Nature. 2021; 591: 639-644Crossref PubMed Scopus (1064) Google Scholar Here, we generated a set of novel, potently SARS-CoV-2 neutralizing monoclonal antibodies and investigated the role of SHM in the enhancement of their affinity and neutralization breadth. To this end, we used blood samples of COVID-19 convalescent individuals 9–10 months after recovery from the disease. Importantly, the blood donors were infected during the first wave of the pandemic in Germany, in March and April 2020, before new variants of concern (VOC) emerged. In three independent rounds (MB021, MB025 & MB027) IgD- IgM- class-switched B cells were enriched for spike- and/or RBD-specific MBC from the blood samples (Fig. 1a). Pairs of immunoglobulin heavy- and light-chain V(D)J-regions were sequenced from single MBC and recombinant mAbs were produced. Only RBD-specific mAbs exerted virus neutralizing activity (Fig. 1b). For each neutralizing mAb a germline version was created by identification and reversion of the VH/VL SHM (Table S1). As expected, SHM enhanced the binding and neutralization of most mAbs (Fig. 1c). Interestingly, the mAb MB025_A09L exerted very similar binding and neutralizing potency in the germline and the hypermutated version (Fig. 1d). Questioning what effect other than increasing the affinity may be conferred by increased numbers of SHM, we compared germline and hypermutated mAbs with respect to their neutralization of SARS-CoV-2 VOC. The mAbs MB025_A07K and MB025_A09L required SHM for the neutralization of the Alpha and Delta variants, respectively. In the already broadly neutralizing mAb MB027_E04L, SHM primarily enhanced the affinity, but not the breadth (Fig. 1e, full panel in Fig. S1). A plethora of antibody-antigen structures have been published in the last few years and deposited in databases.4Raybould M.I.J. Kovaltsuk A. Marks C. Deane C.M. CoV-AbDab: the coronavirus antibody database.Bioinformatics. 2021; 37: 734-735https://pubmed.ncbi.nlm.nih.gov/32805021/Crossref PubMed Scopus (197) Google Scholar We hypothesized that this data resource can be used to infer the binding mode of the antibodies identified in this study. Comparison of all PDB entries with the V(D)J sequences of the antibodies identified above revealed multiple homologs for the mAbs MB025_A07K, MB025_A09L and MB027_D06L, which qualified them to serve as templates (Fig. 2a). Via homology-directed molecular docking, we revealed the most likely binding pose of the three antibodies to the RBD of the spike protein, except for MB025_A09L, where only the germline version was docked in a manner comparable to the template (Fig. 2b). The generated in silico models indicated that the light chain CDR1 of MB025_A07K was located in close proximity to the residue N501 of the spike protein. Antibody docking to the RBD of the Wildtype and the Alpha variants revealed that the germline light chain CDR1 was unable to make contact to the Alpha variant RBD, while the three hypermutations that were located within the CDR1 facilitated RBD binding despite of the presence of the N501Y mutation (Fig. 2c). In conclusion, by reverting the SHM in SARS-CoV-2 neutralizing mAbs back to germline, we demonstrated that affinity maturation in MBC not only enhances the binding strength of antibodies, but also modulates their binding to enhance neutralization of future virus variants. Certain germline gene elements such as VH3–53 dominate B cell responses against SARS-CoV-2 and public clonotypes that use VH3–53 have been identified that are found in multiple unrelated individuals.5Andreano E. Rappuoli R. Immunodominant antibody germlines in COVID-19.J Exp Med. 2021; 218e20210281Crossref PubMed Google Scholar, 6Yuan M. Liu H. Wu N.C. Lee C.-C.D. Zhu X. Zhao F. et al.Structural basis of a shared antibody response to SARS-CoV-2.Science. 2020; 369: 1119-1123Crossref PubMed Scopus (367) Google Scholar In accordance with these findings, mAbs isolated from MBC early after recovery are close to the germline, but already potently neutralized.7Kreer C. Zehner M. Weber T. Ercanoglu M.S. Gieselmann L. Rohde C. et al.Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients.Cell. 2020; 182: 843-854.e12Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar We observed that VOC can escape from neutralization by artificial germline versions of mAbs, while the matured versions neutralize VOC. This effect is particularly relevant for booster vaccinations that are applied after a prolonged period of time because under such conditions pre-existing MBC could be re-engaged in secondary plasmablast responses to release matured antibodies into the serum. Indeed, the neutralization breadth markedly improves after an mRNA boost.8Muecksch F. Wang Z. Cho A. Gaebler C. Ben Tanfous T. DaSilva J. et al.Increased memory B cell potency and breadth after a SARS-CoV-2 mRNA boost.Nature. 2022; 607: 128-134Crossref PubMed Scopus (153) Google Scholar An alternative explanation that was proposed is the masking of immunodominant epitopes by pre-existing plasma antibodies.1Inoue T. Kurosaki T. Memory B cells.Nat Rev Immunol. 2023; (online ahead of print)https://www.nature.com/articles/s41577-023-00897-3.pdfCrossref PubMed Scopus (15) Google Scholar, 9Meyer-Hermann M. Injection of antibodies against immunodominant epitopes tunes germinal centers to generate broadly neutralizing antibodies.Cell Rep. 2019; 29: 1066-1073.e1065Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Both mechanisms are not mutually exclusive and might simultaneously apply, depending on the actual antibody titers. One potential shortcoming of our methodology is that SHM can reliably only be identified within the VH and VL gene segments, while the germline sequence of the CDR3 is often impossible to reconstruct. Earlier studies that investigated the effect of antibody maturation involved longitudinal sampling and detection of the same clonotype in early and late samples.3Gaebler C. Wang Z. Lorenzi J.C.C. Muecksch F. Finkin S. Tokuyama M. et al.Evolution of antibody immunity to SARS-CoV-2.Nature. 2021; 591: 639-644Crossref PubMed Scopus (1064) Google Scholar This is a rather demanding approach, whereas deduction and generation of germline versions, as we did, can be applied to every human mAb sequence. This allows to study even rare clones, that would otherwise be missed. We propose a mechanistic explanation, how the mAb MB025_A07K gained the ability to neutralize the Alpha variant through molecular docking of in silico predicted structures. Molecular docking of antibody structures is intrinsically error-prone and the derived structures may differ from experimentally determined ones. We reduced the risk of creating artefacts by directing the docking with antibody-antigen interaction data that was derived from highly homologous mAbs with already solved structures. Antibodies, either elicited by infection or vaccination or passively administered as mAbs, are important tools against infectious diseases and novel pandemic viruses such as SARS-CoV-2.10Gruell H. Vanshylla K. Weber T. Barnes C.O. Kreer C. Klein F. Antibody-mediated neutralization of SARS-CoV-2.Immunity. 2022; 55: 925-944Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar However, emerging variants can render these antibodies ineffective, resulting in breakthrough infections and unsuccessful patient treatment.10Gruell H. Vanshylla K. Weber T. Barnes C.O. Kreer C. Klein F. Antibody-mediated neutralization of SARS-CoV-2.Immunity. 2022; 55: 925-944Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Studying the inbuilt function of MBC to anticipate future virus variants may help to develop more effective vaccines and next-generation recombinant mAb products. The statistical analysis for linear and non-linear regression was performed in GraphPad Prism 8.4.2. This study was supported by the following funding.